Reversible protein phosphorylation represents the cellular basis for integration of key signaling pathways, mediating a fine crosstalk between external effector molecules and intracellular events. In the heart, Ca2+ cycling and contractility are controlled by a fine balance of protein kinase and phosphatase activities, in response to various second messenger signals.
Demands on the heart's pumping action, during fight-or-flight situations, can increase human cardiac output by nearly 5-fold, and this is linked to β-adrenergic activation of the camp dependent protein kinase (PKA). PKA then phosphorylates a set of key regulatory Ca2+ handling proteins that control excitation-contraction coupling cycle, such as phospholamban, the ryanodine receptor, the L-type channel Ca2+ and troponin I (Bers, D. M., 2002 Nature; 415:198-205).
Although the protein kinases and their phospho-protein substrates, underlying augmentation of the heart's pumping action have been well characterized, similar studies on the protein phosphatases, reversing the increased cardiac contractility are less well developed. Stemming from a common gene family, the major Ser/Thr phosphatases (type 1, type 2A and type 2B (calcineurin), are highly homologous proteins (40-50%) (Cohen, P., 1990 Phosphoprotein Res; 24:230-5) that play critical roles in the control of cardiac contractility and hypertrophy. Overexpression of the catalytic subunit of protein phosphatase 2A has been shown to decrease cardiac function and lead to a pathologic cardiac hypertrophy (Brewis, N. et al., 2000 Am J Physiol Heart Circ Physiol; 279:H1307-18; Gergs, U. et al., 2004 J Biol Chem.). Furthermore, calcineurin, a calcium dependent phosphatase, induces hypertrophy by its regulation of the NFAT transcription factor activity. 5 Interestingly, inhibition of this phosphatase blocks cardiac hypertrophy in vivo and in vitro (Brewis, N. et al., 2000; Molkentin, J. D., 1998 Cell; 93:215-28).
In human and experimental heart failure, the activity of the type 1 phosphatase associated with the sarcoplasmic reticulum (SR) is significantly increased, suggesting that this may be a contributing factor to depressed function, dilated cardiomyopathy and premature death (Huang, B. et al., 1999 Circ Res; 85:848-55; Sande, J. B., et al., 2002 Cardiovasc Res; 53:382-91; Boknik, P. et al., 2000 Naunyn Schmiedebergs Arch Pharmacol; 362:222-31; Gupta, R. C. et al., 1997 Circulation; 96 (Suppl 1): I-361; Neumann, J. 1997 J Mol Cell Cardiol; 29:265-72; Carr, A. N. et al., 2002, Mol Cell Biol; 22:4124-35). However, the role of phosphatase inhibition in β-adrenergic responsiveness was not previously known.